JP2014101362A - Liquid formulation of g-csf conjugates - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide polymer G-CSF that is applied to conjugates, and stable at high temperatures (above refrigerator temperature which is usually 2-8°C).SOLUTION: The invention relates to a liquid pharmaceutical composition comprising granulocyte colony stimulating factor polypeptide conjugated with polymer and having pH value in the range of 4.5-5.5. The composition further comprises a surfactant and, as an arbitrary selection, one or a plurality of other pharmaceutically acceptable excipient. Furthermore, the composition of the invention does not comprise tartaric acid or its salt, or succinic acid or its salt as buffer solution, and does not comprise amino acid as stabilizer. The composition has satisfactory storage stability, and is especially useful for prevention and treatment of disorders and medical application where granulocyte colony stimulating factor preparations are considered as useful remedies.

Description

  The present invention relates to a liquid pharmaceutical composition comprising a granulocyte colony stimulating factor polypeptide conjugated to a polymer and having a pH value in the range of 4.5 to 5.5. The composition further comprises a surfactant and optionally one or more other pharmaceutically acceptable excipients. Furthermore, the composition of the present invention does not contain succinic acid or a salt thereof unless it contains tartaric acid or a salt thereof as a buffer, and does not contain an amino acid as a stabilizer. The composition exhibits good storage stability and is particularly useful for the prevention and treatment of diseases and medical indications where granulocyte colony stimulating factor formulations are considered useful treatments.

  Granulocyte colony stimulating factor (G-CSF) is a hematopoietic growth factor that stimulates the proliferation of hematopoietic progenitor cells and the activation of differentiated and mature neutrophils. G-CSF can support neutrophil proliferation in vitro and in vivo. The human form of G-CSF was cloned in 1986 by Japanese and American groups (Non-patent Document 1). Natural human glycoproteins exist in two forms, one having 174 amino acids and the other having 177 amino acids. The more abundant and more active 174 amino acid form has been used in pharmaceutical development by recombinant DNA technology.

  Large quantities of recombinant G-CSF are produced in recombinant Escherichia coli and have been successfully used in clinical applications to treat cancer patients suffering from chemotherapy-induced neutropenia. G-CSF produced by E. coli is a polypeptide chain consisting of 175 amino acids containing one extra methionine at the N-terminus. This protein is produced by expressing the G-CSF gene in E. coli and purifying the protein product. Such protein products are hydrophobic proteins with 5 cysteine residues, 4 of which are involved in disulfide bonds. One free cysteine residue is usually involved in the formation of higher molecular weight aggregates upon storage of the solution. Protein aggregates can also be formed from oxidized forms of proteins that result from oxidation of internal methionine residues in the main sequence of the protein. Of the four methionine residues, one is located at the N-terminus and the other three are located internally. The oxidized form of the protein containing oxidized methionine at position 122 can be separated from the form containing oxidized methionine at position 127 or 138 and the native protein by standard reverse phase HPLC separation (such position is 175). (Calculated against methionyl-G-CSF consisting of amino acids).

  Recombinant human G-CSF synthesized in an E. coli expression system is called filgrastim (international general name, INN). The structure of filgrastim is slightly different from the natural glycoprotein. Another type of recombinant human G-CSF is called lenograstim (INN), which is synthesized in Chinese hamster ovary (CHO) cells. Filgrastim and Lenograstim are traded in Europe under the trade names Neupogen® and Granocyte, respectively.

However, the above commercially available recombinant human G-CSF has a short medicinal effect and often has to be administered at least twice a day during the leukopenia state. A molecule with a long circulating half-life will reduce the number of doses needed to alleviate leukopenia and, as a result, prevent infectious diseases. Another problem with currently available recombinant human G-CSF products is the occurrence of dose-dependent bone pain. Since bone pain is experienced by patients as a significant side effect of treatment with recombinant human G-CSF, a dose that is essentially free of such effects or does not cause or at least reduce bone pain It would be desirable to provide a recombinant human G-CSF product that is effective in Therefore, there is a clear need for formulations comprising improved recombinant G-CSF molecules and preparations that are ready for stable use.

For example, Patent Documents 1 to 11 report protein engineering variants of human G-CSF in G-CSF variants.
Modifications have also been reported that introduce at least one additional carbohydrate chain into human G-CSF and other polypeptides as compared to native polypeptides (US Pat. No. 6,057,049). Furthermore, polymer modifications of native human G-CSF involving attachment of poly (ethylene glycol) (PEG) groups have been reported and studied (Patent Documents 13-17).

  It is generally accepted that when these proteins are conjugated to a polymer molecule, the stability of the protein is improved and the immune response to these proteins is reduced. Patent Document 18 shows that biogenic protein modified with PEG exhibits immunogenicity and antigenicity, and circulation in the bloodstream is considerably longer than that of unbound protein, that is, the clearance rate is reduced. Yes.

  Attachment of synthetic polymers to peptide backbones to improve the pharmacokinetic properties of glycoprotein therapeutics is well known in the art. An exemplary polymer attached to peptides is PEG. The use of PEG to derivatize peptide therapeutics has been demonstrated to reduce the peptide's immunogenicity. For example, U.S. Patent No. 6,057,031 discloses an enzyme linked to PEG or poly (propylene glycol) (PPG) and a non-immunogenic polypeptide such as a peptide hormone. In addition to a decrease in immunogenicity, clearance time in the blood circulation is prolonged due to an increase in the size of the PEG conjugate of the polypeptide in question.

  Pegfilfilgrastim (INN) is a covalently linked conjugate of recombinant methionyl human G-CSF (filgrastim) and one 20 kDa monomethoxy-PEG molecule. The monomethoxy-PEG molecule is covalently linked to the N-terminal methionyl residue of filgrastim. Pegfilgrastim is traded in Europe under the trade name Neulasta.

  The basic form of attachment of PEG and its derivatives to a peptide is non-specific coupling through the amino acid residues of the peptide (see, for example, Patent Documents 20 to 24). Another form of attachment of PEG to peptides is by non-specific oxidation of glycoprotein glycosyl groups (see, for example, US Pat.

In these non-specific methods, PEG is added in a random non-specific manner to reactive residues on the peptide backbone. Random addition of PEG molecules has drawbacks, including that the final product lacks homogeneity and may reduce the biological or enzymatic activity of the peptide. Over the last few years, more site-specific methods have been developed for attaching synthetic polymers or other labels to peptides, enabling the production of homogeneous peptide therapeutics specifically bound by the action of enzymes in vitro. It turns out. Conjugates based on these enzymes have the advantage of regioselectivity and stereoselectivity. The two main classes of enzymes used in the synthesis of binding peptides are glycosyltransferases (eg sialyltransferases, oligosaccharyltransferases, N-acetylglucosaminyltransferases) and glycosidases. These enzymes can be used for the specific attachment of sugars that can later be modified to have a therapeutic moiety. Alternatively, glycosyltransferases and modified glycosidases may be used to convert directly modified sugars into peptide backbones (see, for example, US Pat. A method of combining a chemical synthesis component and an enzymatic synthesis component is also known (see, for example, Patent Document 29).

  Various methods of conjugating polypeptides such as G-CSF, polymer moieties such as PEG are well known and extensively described in the prior art. A sugar-PEGylated G-CSF preparation is described in Patent Document 10, for example. Another patent application relating to the preparation of conjugates between G-CSF and PEG moieties is US Pat. In this method, the conjugate is linked by an intact glycosyl linking group interposed therebetween and covalently attached to the G-CSF polypeptide and the modifying group. The conjugate is formed from both a glycosylated G-CSF polypeptide and a non-glycosylated G-CSF polypeptide by the action of a glycosyltransferase. Glycosyltransferases ligate modified sugar moieties on amino acids or glycosyl residues on the polypeptide. The disclosures of patent document 10 and patent document 11 are explicitly cited in connection with the present invention.

  In addition to PEG, other polymer moieties have been described as useful conjugates with G-CSF and other therapeutic agent proteins. U.S. Patent No. 6,057,089 shows a biocompatible protein polymer compound produced in particular by the binding of a biologically active protein with a biocompatible polymer derivative. The biocompatible polymer used is a highly reactive branched polymer, and the resulting conjugate comprises a long linker between the polymer derivative and the protein. According to Patent Document 32, examples of biocompatible polymers include PEG, PPG, polyoxyethylene (POE), polytrimethylene ethylene glycol, polylactic acid and derivatives thereof, polyacrylate esters and derivatives thereof, polyamino acids, polyurethanes, Water-soluble polymers such as polyphosphazenes, poly (L-lysine), polyalkylene oxides (PAO), polysaccharides, dextrans, and immunogenic polymers such as polyvinyl alcohol and polyacrylamide are included.

  US Pat. No. 6,057,033 describes a biocompatible polymer formed by covalently bonding a biologically inert polymer or polymer derivative to a pharmaceutically pure synthetic hydrophilic polymer via a specific type of chemical bond. A conjugate is disclosed. Natural polymers and derivatives thereof include proteoglycans such as hyaluronic acid, chondroitin sulfate A, B, and C, chitin, heparin, heparin sulfate, dextran such as cyclodextran, lipids such as hydroxyethyl cellulose, cellulose ether and starch, and triglyceride , And phospholipids are disclosed.

Patent Document 15 describes an N-terminal chemically modified protein compound and a production method thereof. In particular, it describes a G-CSF composition produced by attaching a water-soluble polymer to the N-terminus of G-CSF. Examples of water-soluble polymers listed in Patent Document 15 are copolymers of ethylene glycol and propylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane. , Ethylene / maleic anhydride copolymers, polyamino acids (which can be either homopolymers or random copolymers), poly (n-
Vinylpyrrolidone) polyethylene glycol, PPG homopolymer, polypropylene oxide / ethylene oxide copolymer, or polyoxyethylenated polyol.

U.S. Patent No. 6,057,031 describes a polypeptide conjugate with reduced allergenicity, comprising a polymer carrier molecule to which two or more polypeptide molecules are bound. These conjugates are produced by activating the polymer carrier molecule, reacting two or more polypeptide molecules with the activated polymer carrier molecule, and blocking the remaining active groups on the conjugate. . U.S. Patent No. 6,057,031 lists various polymer carrier molecules including natural or synthetic homopolymers such as polyols, polyamines, polycarboxylic acids and heteropolymers containing at least two different attachment groups. Examples include star PEG, branched PEG, polyvinyl alcohol, polycarboxylate, polyvinyl pyrrolidone and poly-D, L-amino acid. The conjugate of Patent Document 34 includes dextran such as carboxymethyldextran, cellulose such as hydroxyethyl cellulose or hydroxypropyl cellulose, hydrated solution of chitosan, hydroxyethyl starch or hydroxypropyl starch, glycogen, agarose, guar gum, inulin, pullulan, Also includes xanthan gum, carrageenan, pectin, alginic acid and the like.

  Patent Document 35 relates to a method for binding a protein to a starch-derived modified polysaccharide. The binding action between a protein and a polysaccharide (ie hydroxyalkyl starch) is a covalent linkage formed between the terminal aldehyde group resulting from chemical modification of the terminal aldehyde group of the hydroxyalkyl starch molecule and the functional group of the protein. It is. An amino group, a thio group, and a carboxy group are disclosed as reactive groups of the protein.

  US Pat. No. 6,053,097 is a preparation of a conjugate of hydroxyalkyl starch (HAS) and G-CSF protein, wherein at least one functional group of the polymer or derivative thereof reacts with at least one functional group of the protein, thereby The preparation of conjugates forming covalent linkages is described. Other prior art documents relating to HAS, preferably HES, of polypeptides are US Pat.

  Although several approaches have been described in the prior art to modify such therapeutic polypeptide with a polymer moiety in order to extend the clearance time of therapeutic polypeptide such as G-CSF and reduce immunogenicity. There appears to be little research on the development of such advantageous formulations of polymer-G-CSF conjugates.

  The aforementioned Neulasta® product is a liquid composition for subcutaneous injection. The formulation contains pegfilgrastim, sodium acetate, sorbitol, polysorbate 20 and water for injection and has a pH of 4.0 (see http://www.neulasta.com and ROTE LISTE 2007). Neulasta® and Neupogen® products (both sold by Amgen) are nearly identical in terms of buffer, excipient and solution pH values. Neupogen® includes filgrastim (instead of pegfilgrastim), sodium acetate, sorbitol, polysorbate 80 and water for injection and has a pH of 4.0 (http: //www.neupogen. com and ROTE LISTE 2007).

  The present invention relates to a liquid pharmaceutical composition comprising a polymer G-CSF conjugate specifically developed to take into account the properties of the polymer G-CSF conjugate. Although several approaches have been reported in the prior art for formulations containing unbound G-CSF, little is known about useful formulations of polymeric G-CSF conjugates.

  Several pharmaceutical compositions developed for unbound G-CSF are presented in the patent literature, which report to encompass formulations in which unbound G-CSF is replaced with PEG-G-CSF conjugates. However, it is clear that we are preparing and testing only for non-G-CSF.

  For example, U.S. Patent No. 6,057,049 describes a stable pharmaceutical composition comprising G-CSF, where the composition has a pH value greater than 4.0 and further comprises an acid but no surfactant. The pharmaceutical composition described in Patent Document 42 has been clearly developed against unbound G-CSF, and the specification includes G chemically modified with PEG or the like that exhibits the same or improved biological activity. -It is stated to include CSF.

Another example is U.S. Patent No. 6,057,034, which also relates to a stable aqueous composition containing G-CSF. The composition comprises succinic acid or tartaric acid or a salt thereof as a buffer and has a preferred pH in the range of 4.0-5.8. According to the specification, G-CSF proteins may be modified synthetically, for example by enzymatic glycosylation or chemical PEGylation.

  U.S. Patent No. 6,057,034 discloses a stable protein formulation containing tryptophan as a stabilizer. The list of proteins includes G-CSF and G-CSF chemically modified with PEG or its equivalent. The G-CSF preparation preferably has a pH of 5-7, more preferably a pH of 6.0-6.7.

  Another example is U.S. Patent No. 6,057,097, which relates to an injectable pharmaceutical preparation having a pH of 6.5-7.4 comprising a bioactive protein as an active ingredient and at least one sugar as a smoothing agent. It is described. Even in this case, G-CSF chemically modified with PEG or an equivalent thereof is also included.

  U.S. Patent No. 6,057,049 describes a G-CSF solution formulation containing at least one amino acid or salt thereof, preferably methionine as a stabilizer. The G-CSF solution formulation preferably has a pH of 5-7, more preferably a pH of 5.5-6.8. Again, G-CSF chemically modified with PEG or its equivalent is also included.

  However, none of the preparations disclosed in the prior art are preparations of specific polymer G-CSF conjugates. On the contrary, the solutions described in the patent literature have been developed and tested only for non-G-CSF.

International Publication No. 01/87925 European Patent Application Publication No. 0 456 200A US Pat. No. 6,166,183 US Pat. No. 6,004,548 US Pat. No. 5,580,755 US Pat. No. 5,582,823 US Pat. No. 5,675,941 US Pat. No. 5,416,195 US Pat. No. 5,399,345 International Publication No. 2005/055946 International Publication No. 2006/074467 US Pat. No. 5,218,092 US Pat. No. 5,824,778 US Pat. No. 5,824,784 International Publication No. 96/11953 International Publication No. 95/21629 International Publication No. 94/20069 International Publication No. 94/28024 U.S. Pat. No. 4,179,337 U.S. Pat. No. 4,088,538 US Pat. No. 4,496,689 US Pat. No. 4,414,147 US Pat. No. 4,055,635 International Publication No. 87/00056 International Publication No. 94/05332 US Pat. No. 6,399,336 US Patent Application Publication No. 2003/0040037 US Patent Application Publication No. 2004/0132640 US Patent Application Publication No. 2004/0137557 US Patent Application Publication No. 2004/0126838 US Patent Application Publication No. 2004/0142856 International Publication No. 02/09766 International Publication No. 94/01483 International Publication No. 97/30148 International Publication No. 03/074087 International Publication No. 2005/014050 International Publication No. 2005/014655 International Publication No. 2005/092390 International Publication No. 2007/031266 International Publication No. 2005/092928 International Publication No. 2005/092391 International Publication No. 2005/042024 International Publication No. 2005/039620 European Patent Application Publication No. 1 260 230 A1 European Patent Application Publication No. 1 336 410 Al European Patent Application Publication No. 1 329 224 Al

Nagata et al. (1986) Nature 319: 415-418

  The problem underlying the present invention is to provide a polymer G-CSF that is compatible with the conjugate and is stable at high temperatures (usually higher than refrigerators at 2-8 ° C). Furthermore, it is an object of the present invention to provide a pharmaceutical composition that does not require reconstitution at any stage of preparation and causes as little inflammation as possible when administered to a patient.

  These problems are solved according to the present invention by providing an aqueous pharmaceutical composition comprising a polymeric G-CSF conjugate and having a pH in the range of 4.5 to 5.5. The aqueous preparation of the present invention comprises a surfactant and optionally one or more other pharmaceutically acceptable excipients. In a preferred embodiment, the composition does not contain amino acids or derivatives or salts thereof as stabilizers and does not contain tartaric acid or salts thereof or succinic acid or salts thereof as buffers.

  Formulating a composition of polymer G-CSF conjugate having a pH value in the range of 4.5 to 5.5, preferably 5.0, prevents acid hydrolysis of the conjugate bond. It turned out to be surprising. This pH range improves the stability of the solution above the refrigerator temperature (2-8 ° C.), especially at room temperature (ie below 25 ° C.) and even at higher temperatures (eg 40 ° C.). This means that the composition can be stored for an extended period of time without significantly compromising activity and without significantly reducing quality.

  Moreover, apart from storage stability, the composition of the present invention has advantages over a comparative composition having a pH of 4.0. This is because less acidic compositions cause less inflammation when administered to patients.

Unless otherwise defined, the following definitions are set forth to illustrate and explain the meaning and scope of the various terms used to describe the invention herein.
The term “polymer G-CSF” refers to a conjugate between a G-CSF polypeptide and a polymer, wherein a conjugate is formed by a covalent linkage between a functional group of the polymer and a functional group of the polypeptide. . The conjugate may include one or more polymer moieties.

  The term “G-CSF” (or G-CSF polypeptide or G-CSF protein or G-CSF peptide) refers to the in vivo biological activity of native human G-CSF (ie, a protein that can stimulate the differentiation and proliferation of hematopoietic progenitor cells). It refers to the protein it has. G-CSF can be obtained by the assay described in Stute, N., et al. "Pharmacokinetics of subcutaneous recombinant human granulocyte colony- stimulating factor in children" (1992) Blood 79 (11), pages 2849-2854. Can be clearly confirmed.

  In an exemplary embodiment, G-CSF has the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, which represents the wild-type amino acid sequence of human methionyl-G-CSF produced in E. coli, SEQ ID NO: 2 Indicates the amino acid sequence of human G-CSF produced in mammalian cells (eg CHO cells). SEQ ID NO: 1 is a 175 amino acid variant in which the first amino acid is methionine and Thr 134 has a threonine residue. Since SEQ ID NO: 2 is a 174 amino acid variant having the same sequence as the 175 amino acid variant except that there is no leading methionine, the sequence starts with T and has a threonine residue at position 133.

SEQ ID NO: 1
MTPLGPASSLPQSFLKLKLEQVQQQQGAGAQQKLCTYKYKLCHPEELVLLGHHSLGIPWAPLSSCPSQALQLAGCLSQLHSGLFLYQGLL
SEQ ID NO: 2
TPLGPASSLPQSFLLKLKLEQVRKIQQGGAALQEKLCATYKLCHPEELVLLGHSLGIPRWALSLSPS
One skilled in the art will readily appreciate that the present invention is not limited to the sequences shown herein, but includes variants of G-CSF. Such variants are well known in the art and include one or more amino acid deletions, substitutions or additions to the amino acid sequence set forth above while maintaining the biological activity of native G-CSF. obtain. By way of example, but not at all, G-CSF variants are described in WO 01/87925, EP 0 456 200 A, US Pat. No. 6,166,183, US Pat. No. 6,004,548, US Patent 5,580,755, US Patent 5,582,823, US Patent 5,675,941, US Patent 5,416,195, US Patent 5,399,345, International Publication No. 2005/055946 And International Publication No. WO 2006/074467, which do not limit the application as such.

  The G-CSF polypeptide may or may not be glycosylated. In a preferred embodiment, the G-CSF polypeptide is recombinant human G-CSF produced in E. coli, that is, recombinant human G-CSF having the amino acid sequence shown above in SEQ ID NO: 1 or a variant thereof. It is.

  The polymer can be any polymer that can be covalently linked to a G-CSF polypeptide, and that when covalently linked to a G-CSF polypeptide yields a therapeutically useful polymer G-CSF conjugate. Also good. Some suitable polymers have already been mentioned in the introductory part, including poly (alkylene ethylene glycol) such as PEG and PPG, hydroxyalkyl starch such as hydroxyethyl starch (HES), and international for polymer polymer polypeptides Polymers described in WO 02/09766, WO 96/11953, WO 97/30148. In a preferred embodiment, the polymer is PEG.

The details of the mg / ml concentration used below for the conjugate all relate to the G-CSF moiety only. By definition, the PEG moiety does not consider mass concentration.
While filgrastim has a molecular weight of about 18-19 kD, pegfilgrastim is much larger due to its monomethoxy-PEG moiety and has a molecular weight of about 39 kD. The polymer G-CSF conjugate of the present invention may have a molecular weight in the range of 20-60 kD, preferably in the range of 35-45 kD.

  Suitable PEGs are disclosed in the prior art, for example WO 2005/055946, WO 2006/074467 and WO 01/87329. The PEG moiety may be straight or branched and may have a size of 5-40 kD. Preferably the PEG moiety has a molecular weight of 15-25 kD, most preferably about 20 kD.

  Methods for producing polymer G-CSF conjugates are also described in the prior art. The above mentioned documents relating to the preparation of conjugates between the polypeptide and the polymer part are incorporated. In the present application, the disclosure of these documents, that is, the conjugate described in the present specification and the production method thereof are incorporated.

  Other polymer G-CSF conjugates useful in the present invention are disclosed in WO 96/11953, EP 822 199A, WO 01/51510, WO 2006/0128460, European patent. EP-A 921 131 A and EP-A 744 409. In this application, reference is made explicitly to the disclosure of these documents, namely the conjugates described herein and the methods of making them.

  Those skilled in the art can easily include not only conjugates in which a polymer such as PEG or HES is directly attached to the amino acid residues of the protein, but also conjugates in which the polymer moiety and the G-CSF polypeptide are linked together by a linker. Will understand. For example, a glycosyl group sandwiched between a polypeptide and a PEG moiety is a useful linker within the conjugate of the invention. WO 2006/074467 describes a polymer G-CSF in which a G-CSF polypeptide and a polymer moiety are linked via a glycosyl linker or non-glycosyl linker (eg, substituted or unsubstituted alkyl or substituted or unsubstituted heteroalkyl). A CSF conjugate is described. The disclosure of WO 2006/074467 is incorporated herein by reference.

  The term “PEGG-CSF” (PEGylated G-CSF) refers to a G-CSF protein covalently linked to one or more polyethylene glycol moieties as described below. The PEG group and the G-CSF protein may be linked directly to each other or may be linked via a linker (eg, a glycosyl linker).

In one embodiment of the invention, the polymeric G-CSF peptide conjugate is prepared by the method described in WO 2006/074467. In a preferred embodiment, the polymeric G-CSF peptide is a PEG-G-CSF conjugate having a glycosyl linking group between the PEG modifying moiety and the G-CSF polypeptide. Such a conjugate is referred to as “sugar PEGylated” G-CSF.

  In an illustrative embodiment, a “glycoPEGylated” G-CSF molecule of the invention is a G-CSF peptide that may or may not be glycosylated and an enzyme that has a poly (ethylene glycol) moiety in its structure. Produced by enzyme-mediated formation of conjugates between transposable saccharyl moieties. The PEG moiety may be attached directly to the saccharyl moiety (ie, by forming one group by reaction of two reactive groups) or via a linker moiety (eg, substituted or unsubstituted) The glycosyl linking group may be a sialic acid moiety derivatized with PEG.

  In a preferred embodiment of the invention, the glycosyl linker is attached to the G-CSF protein via O-glycosylation, preferably via O-glycosylation at the position of the threonine residue of the G-CSF protein. .

The glycosyl linker preferably comprises a monosaccharide, disaccharide or oligosaccharide, more preferably the glycosyl linker comprises sialic acid and N-acetylgalactosamine.
In one embodiment of the invention, the polymeric G-CSF peptide conjugate comprises the following moieties:

In the formula, R ₁ is a moiety containing a linear or branched poly (ethylene glycol) residue, and L
Is a linker that is a member selected from a bond, substituted or unsubstituted alkyl, and substituted or unsubstituted heteroalkyl, the peptide is glycosylated with a glycosyl group, and the moiety is covalently linked to the glycosyl group . The glycosyl group is preferably N-acetylgalactosamine. In a preferred embodiment, the G-CSF peptide is glycosylated at a threonine residue, preferably a threonine residue at position 134 (calculated for a methionyl-G-CSF polypeptide having a total of 175 amino acids with the N-terminal group methionine). Is done. In a preferred embodiment, the threonine residue is a linear poly (ethylene glycol) group and L is heteroalkyl.

The above G-CSF peptide conjugate is
(A) The substrate G-CSF peptide can be produced by a method comprising contacting a PEG sialic acid donor having the formula:

Wherein R ₁ and L are as defined above and are enzymes that can transfer the PEG sialic acid moiety from the donor onto the glycosyl group of the substrate G-CSF peptide. In a preferred embodiment, the enzyme is a sialyltransferase, for example STβGalNAcI as described in WO 2005/055946.

The above G-CSF peptide conjugate is
(A) contacting a non-glycosylated substrate G-CSF peptide with a glycosyl donor and an enzyme capable of transferring a glycosyl group moiety from the donor onto the substrate G-CSF peptide; and (b) a glycosylated G-CSF peptide, It can be produced by a method comprising contacting with a PEG sialic acid donor having the formula:

Wherein R ₁ and L are as defined above and are enzymes that can transfer the PEG sialic acid moiety from the donor onto the glycosyl group of the substrate G-CSF peptide. Steps (a) and (b) may be sequential reactions or simultaneous reactions. In a preferred embodiment, the glycosyl donor is UDPN-acetylgalactosamine. In a preferred embodiment, the enzyme of step (a) is N-acetylgalactosaminyltransferase, the enzyme of step (b) is a sialyltransferase, eg, GalNAcT2 in step (a) and STβGalNAcI in step (b). .

G-CSF may be produced by chemical synthesis, may be from any human or another mammalian source, and purified to a natural source such as human placenta, human blood or human urine You may get it from the source. In addition, many epithelial cancers, acute spinal leukemia cells and various tumor cell lines can express this factor.

  Preferably, G-CSF is produced by genetic recombination. This includes the expression of exogenous DNA sequences obtained by cloning of the genome or cDNA or by DNA synthesis in eukaryotic or prokaryotic hosts. Suitable prokaryotic hosts include a variety of bacteria including the preferred host E. coli. Suitable eukaryotic hosts are S. cerevisiae. yeast and Chinese hamster ovary (CHO) cells such as cerevisiae and mammalian cells such as monkey cells.

Recombinant production methods for proteins such as G-CSF are well known in the art. In general, this involves transfection of the host cell with an appropriate expression vector, culturing the host cell under conditions that allow production of the protein, and purification of the protein from the host cell. For more information, see, for example, Souza, LM et al. 1986, Recombinant human granulocyte colony-stimulating factor: effects on normal and leukemic myeloid cells, Science (1986) 232: 61-65; Nagata, S. et al. 1986, Molecular cloning and expression of cDNA for human granulocyte colony- stimulating factor, Nature (1986) 319: 415-418; Komatsu, Y. et al. 1987, Cloning of granulocyte colony-stimulating factor
See cDNA from human macrophages and its expression in Escherichia coli, Jpn. J. Cancer Res. (1987) 78: 1179-1181.

  In a preferred embodiment, the G-CSF has the amino acid sequence of human mature G-CSF (see Nagata, S. et al. (1986), supra), and may further comprise methionine at the end of the net; The result is a protein consisting of 175 amino acids (see SEQ ID NO: 1 above). Furthermore, instead of methionine, G-CSF may contain serine or threonine residues.

  The protein is then purified according to conventional downstream processing protocols. Purification methods suitable for G-CSF include, for example, WO 87/01132, European Patent Application Publication No. 0 719 860A, European Patent Application Publication No. 1 458 757 A, and European Patent Application Publication No. 1 527 188A. , WO 03/051922, WO 01/04154, and WO 2006/097944.

  In one embodiment of the invention, a polymeric G-CSF peptide conjugate is prepared as described in Example 1 provided herein. This conjugate is characterized in that the G-CSF polypeptide and the PEG moiety are linked through N-acetylgalactosaminyl (GalNAc) and sialic acid (SA) groups. The conjugate has the following G-CSF-GalNAc-SA-PEG structure.

Where R ₁ and L are as defined above and AA is an amino acid residue of G-CSF.
In an exemplary embodiment, R ₁ is a linear PEG moiety, as shown below, attached to the G-CSF polypeptide via a sialic acid group and a GalNAc group.

In the formula, L is as defined above, AA is an amino acid residue of G-CSF, and f is an integer selected from 1 to 2500.
In certain embodiments, the polymeric G-CSF conjugate has the following formula:

In the formula, AA is threonine 133 of G-CSF (threonine 134 when N-terminal methionine is present), and f is an integer selected from 1 to 2500.
The preparation of the present invention is a liquid composition (eg, an aqueous solution). For injection purposes, it is preferred to use pure water as the solvent. However, other conventional solvents suitable for pharmaceutical formulations can also be used. In a preferred embodiment of the invention, the pharmaceutical composition is an isotonic solution.

Furthermore, there is no need for reconstitution at any stage in the preparation of the liquid solution formulation of the present invention. A solution is a preparation that is ready for use.
The pharmaceutical composition of the present invention has a pH in the range of 4.5 to 5.5. In a preferred embodiment, the pH value is between 4.7 and 5.3, more preferably between 4.8 and 5.2, most preferably between 4.9 and 5.1.

  If adjustment is necessary to achieve the desired pH range, the pH value is adjusted with an appropriate solution; an acidic solution when a decrease in pH value is indicated, an alkaline solution when an increase in pH value is indicated In solution. Suitable acidic solutions are, for example, hydrochloric acid, phosphoric acid, citric acid and sodium hydrogen phosphate and potassium hydrogen phosphate. Suitable alkaline solutions include alkali and alkaline earth metal hydroxides, alkali carbonates, alkali acetates, alkali citrates, and dialkali hydrogen phosphates (eg, sodium hydroxide, sodium acetate, sodium carbonate, citric acid). Sodium, disodium, potassium phosphate, ammonia).

Preferably, the pH of the solution is adjusted using sodium hydroxide. As a result, the formulations of the present invention may contain sodium ions. Sodium is present at a concentration of less than 10 mmol / l, usually less than 6 mmol / l.

  The formulations of the present invention include one or more surfactants. Typical examples of surfactants include: nonionic surfactants such as sorbitan fatty acid esters such as sorbitan monocaprylate, sorbitan monolaurate, and sorbitan monopalmitate; glyceryl monocaprylate; Glycerin fatty acid esters such as glyceryl monomyristate and glyceryl monostearate; polyglycerin fatty acid esters such as decaglyceryl monostearate, decaglyceryl distearate, and decaglyceryl monolinoleate; sorbitan polyoxyethylene monolaurate, polyoxyethylene Sorbitan monooleate, sorbitan polyoxyethylene monostearate, sorbitan polyoxyethylene monopalmitate, sorbitan polyoxyethylene trioleate, and Polyoxyethylene sorbitan fatty acid esters such as sorbitan reoxyethylene tristearate; Polyoxyethylene sorbitol fatty acid esters such as sorbitol polyoxyethylene tetrastearate and sorbitol polyoxyethylene tetraoleate; Polyoxy such as glycerin polyoxyethylene monostearate Ethylene glycol fatty acid ester; polyethylene glycol fatty acid ester such as polyethylene distearate glycol; polyoxyethylene alkyl ether such as polyoxyethylene lauryl ether; polyoxyethylene polyoxypropylene glycol ether, polyoxyethylene polyoxypropylene propyl ether, and polyoxy Polio such as ethylene polyoxypropylene cetyl ether Polyoxyethylene alkylphenyl ethers such as polyoxyethylene nonylphenyl ether; polyoxyethylene hydrogenated castor oils such as polyoxyethylene castor oil and polyoxyethylene hydrogenated castor oil (polyoxyethylene hydrogenated castor oil); Polyoxyethylene wax derivatives such as polyoxyethylene sorbitol wax; polyoxyethylene lanolin derivatives such as polyoxyethylene lanolin; polyoxyethylene fatty acid amides such as polyoxyethylene stearamide having an HLB value of 6-18; anion interface Activators such as alkyl sulfates with C10-18 alkyl such as sodium cetyl sulfate, sodium lauryl sulfate and sodium oleyl sulfate; Polyoxyethylene alkyl ether sulfates having an average EO mole number of 2-4 and C10-18 alkyl such as sodium lauryl sulfate; alkylsulfosuccinic acid ester salts having C8-18 alkyl such as sodium lauryl sulfosuccinate And natural surfactants such as lecithin; glycerophospholipids; sphingophospholipids such as sphingomyelin; sucrose fatty acid esters of C12-18 fatty acids. One or more of these surfactants can be added in combination with the formulations of the present invention.

  Preferred surfactants are alkyl esters of polyoxyethylene sorbitan, preferably polysorbate 20, 21, 40, 60, 65, 80, 81, 85, most preferably polysorbate 20 and 80.

  The concentration of the surfactant in the preparation is usually in the range of 0.0005% (w / v) to 0.05% (w / v), more preferably 0.001% (w / V) to 0.01% (w / v), more preferably 0.002% (w / v) to 0.006% (w / v), most preferably 0.003%. The range is from (w / v) to 0.004% (w / v).

  Typically, the formulations of the invention comprise surfactant polysorbate 20 or 80 at a concentration of 0.003% (w / v), 0.0033% (w / v) or 0.004% (w / v), Polysorbate 20 is preferred.

  The formulations of the present invention include a physiologically acceptable buffer. Suitable buffers are well known in the field of solution formulations, for example (preferably sodium monohydrogen phosphate-sodium dihydrogen phosphate) phosphate buffer, citrate buffer, lactate buffer, acetate buffer, carbonate Buffer, BisTris, MES, and glycine-HCl). The use of an acetate buffer, i.e. the use of acidic acids and their salts (for example alkali or ammonium salts) is preferred.

  The buffer is usually present in the formulation at a concentration of 1-100 mmol / l, preferably 2-50 mmol / l, most preferably 5-20 mmol / l. In a preferred embodiment, the buffer is acetate present at 10 mmol / l, most preferably at 10 mmol / l.

  The concentration of the buffer (eg, acetate) is selected so as to provide a pH stabilizing effect as well as sufficient buffer capacity. At the same time, however, the ion concentration and thus the conductivity of the solution is as low as possible to avoid the formation of aggregates.

In embodiments of the invention, the conductivity of the final solution formulation is less than 1.0 mS / cm, preferably less than 0.8 mS / cm, more preferably less than 0.5 mS / cm.
Furthermore, the formulation preferably does not contain tartaric acid and succinic acid and salts thereof. It is also preferred that the solution is free of HEPES, TES and tricine.

It is also preferred that the formulation of the present invention does not contain sulfate ions.
Furthermore, in a preferred embodiment, the formulation does not contain preservatives. Here, the term “preservative” means a substance which is conventionally used as a preservative for increasing storage stability and has a bactericidal action at a standard concentration. In particular, the formulation does not contain preservatives such as ethyl chloride, benzyl alcohol, p-chloro-m-cresol and dicarboxylic esters of pyrocarboxylic acid and benzalkonium chloride.

  In an embodiment of the invention, the formulation further preferably comprises a polyol, preferably a sugar alcohol, most preferably mannitol or sorbitol as a tonicity modifying agent. Sorbitol is particularly preferred. The amount of sugar such as sorbitol or mannitol is usually 10.0% (w / v) or less based on the total amount of the solution. Preferably, the sugar concentration is 8.0% (w / v) or less, more preferably 6.0% (w / v) or less, and most preferably 5.0% (w / v) or less. In a preferred embodiment, sorbitol is present in an amount of 5.0% (w / v).

Furthermore, the solution formulation of the present invention preferably does not contain a stabilizer selected from amino acids, derivatives and salts thereof, polymer stabilizers and protein stabilizers.
Formulations of the invention comprising a polymeric G-CSF conjugate are usually administered by injection (subcutaneous, intravenous or intramuscular injection) or parenteral routes such as transdermal, mucosal, nasal, or pulmonary administration, There may be.

  The polymer G-CSF conjugate is usually at a concentration of 1.0 to 30.0 mg / ml, preferably 5.0 to 20.0 mg / ml, most preferably 8.0 to 12.0 mg / ml. Present in the formulation. In a preferred embodiment, the polymer G-CSF conjugate is PEG-SA-GalNAc-G-CSF present in an amount of 10.0 mg / ml.

In a preferred embodiment, the formulation comprises polymer G-CSF as an active ingredient, a surfactant, a buffer, a tonicity modifying agent, sodium ions, and water and no other ingredients. Most preferably, the aqueous formulation of the present invention comprises a sugar PEGylated G-CSF as an active agent, at least one of polysorbate 20 and polysorbate 80 as a surfactant, and sorbitol and mannitol as tonicity modifying agents. It contains at least one of them, acetate as a buffer, and sodium, and does not contain other excipients.

  In another aspect of the invention, the aqueous formulation of the invention described above is diluted to obtain a diluted aqueous formulation suitable for use in children. Dilution solutions suitable for the treatment of children are obtained by diluting the above-described solutions of the invention 1: 2 to 1: 8.

  The present invention further relates to a pharmaceutical container comprising the aqueous preparation of the present invention or a diluted solution obtained by dilution from the aqueous preparation. Suitable pharmaceutical containers are understood from the prior art. The container may be, for example, a syringe, glass bottle, infusion bottle, ampoule or carpule. In a preferred embodiment, when the container is a syringe, the syringe is equipped with a needle protection system. Such needle protection systems known from the prior art help to reduce the risk of damage. In another embodiment, the container is a carpul in an injection pen.

  The present invention is further a method for producing the aqueous formulation of the present invention, wherein the polymer G-CSF conjugate as an active agent has a pH in the range of 4.5 to 5.5 and a surfactant. The present invention relates to a method comprising formulating an aqueous formulation comprising a pharmaceutical excipient.

  In another aspect, the invention relates to the use of the aqueous formulation of the invention in the treatment or prevention of neutropenia. Furthermore, the aqueous formulation of the invention can advantageously be used for the treatment or prevention of neurological disorders or for bone marrow transplantation. In general, the pharmaceutical solutions of the present invention are useful for mobilizing stem cells.

  It has been found that the pharmaceutical liquid formulations of the present invention exhibit very good storage stability. Within the scope of the present invention, the term “storage stability” means that the content of active polymer G-CSF still reaches more than 80% of the initial concentration even after storing the formulation at 25 ° C. for 3 months. . Preferably, after 3 months storage at 25 ° C., the remaining content of G-CSF activity reaches at least 85%, more preferably at least 90%, most preferably at least 95% of the initial activity.

  The activity of the polymer G-CSF conjugate can be determined by conventional activity tests as described in the prior art for G-CSF. For example, Draft Monographie "Filgrastim Concentrated Solution" PharmEur. Vol.19, No.l, Jan. 2007, or Stute, N., et al. "Pharmacokinetics of subcutaneous recombinant human granulocyte colony-stimulating factor in children 1" (1992 ) See Blood 79 (11), pages 2849-2854.

In vitro measurement of G-CSF activity was performed by Shirahuji, N. et al. 1989, A new bioassay for human granulocyte colony-stimulating factor (hG-CSF) using murine myeloblasts NFS-60 cells as targets and estimation of its levels in sera from normal healthy persons and patients with infectious and hematological disorders, Exp.
Hematol. (1989) 17, 116-119. The measurement of G-CSF activity in vivo is described in, for example, Tanaka, H. et al. 1991, Pharmacokinetics of recombinant human granulocyte colony- stimulating factor conjugated to polyethylene glycol in rats, Cancer Research (1991) 51,3710-3714. ing. Additional publications describing tests for measuring G-CSF activity include US Pat. No. 6,555,660 and Nohynek, GJ et al. 1997, Comparison of the potency of glycosylated and nonglycosylated recombinant human granulocyte colony- stimulating factors in neutropenic and non-neutropenic CD rats, Cancer Chemother. Pharmacol. (1997) 39, 259-266.

  The purity of the polymer G-CSF conjugate used in the formulation is preferably at least 95%, preferably at least 97%, more preferably at least 99%, most higher than 99%. The degree of purity can be determined by HPLC analysis. Suitable materials and protocols for performing such analyzes can be obtained from commercial suppliers such as Vydac or TOSOH Bioscience (http: // www. Tosohbioscp.de).

Ingredients for formulating the solutions of the present invention can be obtained from conventional suppliers, for example from companies such as Sigma or Merck.
Production of the formulations of the present invention can be carried out by conventional methods. The components of the formulation can be dissolved in an aqueous buffer. Alternatively, the conjugate may be obtained in the form of an aqueous buffer as a result of the purification process.

Finally, the finished liquid formulation is filled into a suitable pharmaceutical container and stored until administered.
The following examples illustrate the present invention without limiting the scope of the invention.
Examples Example 1 Preparation of G-CSF-GalNAc-SA-PEG In the following examples, simultaneous addition of enzymes is used to (a) two intermediate products where each intermediate product is purified before being used in the next step. The preparation of G-CSF-GalNAc-SA-PEG in a method consisting of continuous steps and (b) a method consisting of one step will be described.

a. Preparation of G-CSF-GalNAc (pH 6.2) from G-CSF and UDP-GalNAc using GalNAc-T2 A packaged 3.2 mL buffer of G-CSF (960 μg) was used. Concentrated using an ultrafiltration filter (MWCO 5K) and then reconstituted with 1 mL of 25 mM MES buffer (pH 6.2, 0.005% NaN ³ ). Thereafter, UDP-GalNAc (6 mg, 9
. 24 mM), GalNAc-T2 (40 μL, 0.04 U), and 100 mM MnCl ₂ (40 μL, 4 mM)) were added and the resulting solution was incubated at room temperature.

After 24 hours, MALDI showed that the reaction was complete. The reaction mixture was directly subjected to HPLC purification using elution buffer containing SEC (Superdex 75 and Superdex 200) and PBS (phosphate buffer, pH 4.9 and 0.005% Tween 80). The collection peak of G-CSF-GalNAc was expressed as Centricon 5 KDa.
Concentrate to approximately 150 μL using MWCO filter and adjust volume to 1 ml with PBS (phosphate buffer (pH 4.9 and 0.005% Tween 80). Final protein concentration is 1 mg / mL (A ₂₈₀ ) The yield was 100% and the sample was stored at 4 ° C.

Purification of G-CSF-GalNAc-SA-PEG using purified G-CSF-GalNAc, CMP-SA-PEG (20 KDa) and mouse STOGalN Ac-TI (pH 6.2) G-CSF- containing 1 mg of protein The GalNAc solution was replaced with 25 mM MES buffer (pH 6.2, 0.005% NaN ₃ ), and CMP-SA-PEG (20
KDa) (5 mg, 0.25 μmol) was added. After dissolution, MnCl ₂ (100 μL, 1
00 mM solution) and ST6GalNAc-I (100 μL (mouse enzyme) were added and the reaction mixture was gently shaken for 3 days at 32 ° C. The reaction mixture was concentrated by ultrafiltration (MWCO5K) and 25 mM NaOAc (pH 4. The buffer was exchanged once in 9) and then concentrated to a total volume of 1 mL, after which the product was SP-Sepharose (A: 25 mM NaOAc + 0.005% Tween-80 pH 4.5, retention time 13-18 minutes). , B: 25 mM NaOAc + 0.005% Tween-80 pH4.5 + 2M NaCl) and retention time 8.6 minutes (Superdex 75, flow rate 1 ml / min) SEC (Superdex 75; PBS-pH 7.2, 0.005% Tween 80) The desired fractions were collected, concentrated to 0.5 mL, It was stored at ℃.

b. One-step method One-pot process using mouse SToGalNAc-I (pH 6.0) G-CSF (960 μg protein, dissolved in 3.2 mL product formulation buffer) was reduced to 0 by ultrafiltration (MWCO5K). Concentrated to 5 ml and reconstituted with 25 mM MES buffer (pH 6.0, 0.005% NaN ₃ ) to a total volume of about 1 mL or protein concentration of 1 mg / mL. Then, UDP-GalNAc (6 mg, 9.21 μmol), GalNAc-T2 (80 μL, 80 mU), CMP-SA-PEG (20 KDa) (6 mg, 0, 3 μmol) and mouse enzyme ST6GalNAc-I (120 μL) and 100 mM MnCl ₂ (50 μL) was added. The solution was shaken at 32 ° C. for 48 hours, and SP-Sepharo
Purified using standard chromatographic conditions on se. A total volume of 0.5 ml protein (A ₂₈₀ ) or an overall yield of about 50% was obtained. The structure of the product was converted to MALDI and SDS-
Confirmed by analysis using both PAGEs.

One-pot process using chicken SToGalNAc-I (pH 6.0) 14.4 mg of G-CSF was concentrated to a final volume of 3 mL and 25 mM MES buffer (pH 6).
. The buffer was exchanged with 0, 0.05% NaN ₃ , 0.004% Tween 80) and the volume was adjusted to 13 mL. Then, UDP-GalNAc (90 mg, 150 μmol), GalNAcT2 (0.59 U), CMP-SA-PEG-20KDa (90 mg), chicken ST6GalNAc-I (0.44 U) and 100 mM MnCl ₂ (600 μL)
Was added. The resulting mixture was allowed to stand at room temperature for 60 hours. The reaction mixture was then concentrated using ultrafiltration (MWCO 5K) and centrifugation. The residue (about 2 mL) was dissolved in 25 mM NaOAc buffer (pH 4.5) and concentrated again to a final volume of 5 mL. This sample was used using SP Sepharose, SEC (Superdex 75, 17 minutes, flow rate 0.5 ml / min) and additional SEC (Superdex 200, 23 minutes, flow rate 0.5 ml / min) with a retention time of about 10-23 minutes. Purification gave 3.6 mg (25% overall yield) of G-CSF-GalNAc-SA-PEG-20KDa (A ₂₈₀ and BCA method).

Example 2 Liquid polymer-G-CSF conjugate (PEG-SA-GalNAc-G-CSF) formulation A liquid formulation comprising a sugar PEGylated G-CSF (a conjugate having the structure of PEG-SA-GalNAc-G-CSF) is described below. Were prepared by blending the ingredients in acetate buffer.

The pH value of the composition was adjusted by adding NaOH. All ingredients are European Pharmacopoeia (Ph.E
ur. ) According to the quality.
In addition, the same compositions were prepared having either pH 4.5 or pH 5.5, each with proportionally less or more sodium. A comparative formulation having a pH of 4.0 (as in the Neulasta® formulation) was also formulated.

Example 3 Stability test of formulations according to the invention Compositions of pH 4.5, 5.0 and 5.5 were equally divided into 500 μl / glass bottles and stored at 2-8 ° C. and 25 ° C. After 1, 2, 3, 4.5, 6, 8, 12, and 15 months, the samples were tested for the test parameters given in the table below.

  The predicted specifications for the composition having pH 5.0 were as follows:

  All samples tested at T = 0 month, 1 month, 2 months, 3 months, 4.5 months, 6 months, 8 months, 12 months, and 15 months are predicted specifications Was met. This has been found for all compositions containing sugar PEGylated G-CSF and having a pH of 4.5, 5.0 or 5.5.

  The composition of the present invention is composed of two comparative formulations: Neulasta® (pH 4.0) and a sugar PEGylated G-CSF having a pH of 4.0 (PEG-SA-GalNAc-G-CSF). : Compared with As a result, formulations with higher pH values of 4.5, 5.0 and 5.5 have better storage stability compared to comparative solutions containing sugar PEGylated G-CSF and having a pH of 4.0 It has been found to show. With these collected data, it can be concluded that higher pH values prevent acid hydrolysis of sugar PEG linkages.

  Furthermore, it was observed that the formulations of the present invention have a stability comparable to that of the PEG-G-CSF conjugate known as Neulasta®.

Claims (37)

An aqueous formulation comprising a polymer G-CSF conjugate, having a pH in the range of 4.5 to 5.5, and further comprising a surfactant. The aqueous preparation according to claim 1, wherein the polymer is a polyalkylene glycol. The aqueous preparation according to claim 1 or 2, wherein the polymer and G-CSF are bonded with a glycosyl linker. The aqueous preparation according to claim 3, wherein the linkage by the glycosyl linker is by O-glycosylation. The aqueous formulation of claim 4, wherein the O-glycosylation is located at a threonine residue of a G-CSF protein. The aqueous preparation according to claim 5, wherein the threonine residue is Thr 134 based on the amino acid sequence of methionyl-G-CSF protein or Thr 133 based on the amino acid sequence of natural human G-CSF. The aqueous preparation according to any one of claims 3 to 6, wherein the glycosyl linker comprises a monosaccharide, disaccharide or oligosaccharide. The aqueous preparation according to any one of claims 3 to 7, wherein the glycosyl linker comprises sialic acid and N-acetylgalactosamine. The aqueous preparation according to any one of claims 1 to 8, wherein the surfactant is present at a concentration of 0.0001% (w / v) to 0.05% (w / v). The aqueous preparation according to any one of claims 1 to 9, wherein the surfactant is a polyoxyethylene sorbitan alkyl ester. The aqueous preparation according to claim 10, wherein the polyoxyethylene sorbitan alkyl ester is polysorbate 20 or polysorbate 80. The aqueous preparation according to any one of claims 1 to 11, wherein the pH is in the range of 4.7 to 5.3. The aqueous preparation according to any one of claims 1 to 12, wherein the pH is in the range of 4.9 to 5.1. The aqueous preparation according to any one of claims 1 to 13, further comprising a physiologically acceptable buffer. The aqueous preparation according to claim 14, wherein the buffer contains acetic acid or a salt thereof. The aqueous formulation according to claim 14 or 15, wherein the buffer is present at a concentration of 2 to 50 mmol / l. The aqueous preparation according to any one of claims 1 to 16, further comprising a tonicity modifying agent selected from sorbitol and mannitol. 18. An aqueous formulation according to claim 17, wherein the tonicity modifying agent is present in a concentration of 1-10%. The aqueous formulation according to any one of claims 1 to 18, which does not contain a stabilizer selected from amino acids, polymer stabilizers and protein stabilizers. The aqueous preparation according to any one of claims 1 to 19, which contains no preservative. The aqueous preparation according to any one of claims 1 to 20, which does not contain sulfate ions. The aqueous preparation according to any one of claims 1 to 21, wherein the pH is adjusted using NaOH. The aqueous preparation according to any one of claims 1 to 22, comprising sodium ions. Polymer G-CSF conjugate as active agent, at least one of polysorbate 20 and polysorbate 80 as surfactant, at least one of sorbitol and mannitol as tonicity modifying agent, acetate as buffer, and 24. An aqueous formulation according to any one of claims 1 to 23 comprising sodium. 25. The aqueous formulation according to any one of claims 1 to 24, wherein the polymer G-CSF conjugate is present at a concentration of 1 to 20 mg / ml. 26. The aqueous formulation of any one of claims 1-25, wherein the polymer G-CSF conjugate is present at a concentration of 8-12 mg / ml. 27. A diluted aqueous formulation obtained from the aqueous formulation of any one of claims 1-26, wherein the aqueous formulation is diluted 1: 2 to 1: 8. A pharmaceutical container comprising the aqueous preparation according to any one of claims 1 to 27. The pharmaceutical container according to claim 28, which is a syringe, a glass bottle, an infusion bottle, an ampoule or a carpul. 30. A pharmaceutical container according to claim 28 or 29, which is a syringe provided with a needle protection system. 30. A pharmaceutical container according to claim 28 or 29 which is a carpul in an injection pen. A method for producing the aqueous formulation according to any one of claims 1 to 26, comprising:
A method comprising formulating a polymeric G-CSF conjugate as an active agent into an aqueous formulation having a pH in the range of 4.5 to 5.5 and comprising a surfactant and further a pharmaceutical excipient. 28. An aqueous formulation according to any one of claims 1 to 27 for use in the treatment or prevention of neutropenia. 28. An aqueous formulation according to any one of claims 1 to 27 for use in the treatment or prevention of neurological disorders. 28. An aqueous formulation according to any one of claims 1 to 27 for use in bone marrow transplantation. 28. An aqueous formulation according to any one of claims 1 to 27 for use in stem cell mobilization. 28. An aqueous formulation according to claim 27 for use in pediatric administration.